Search

DMD pathogenic variants for Duchenne and Becker muscular dystrophy are detectable with high sensitivity by standard clinical exome analyses of genomic DNA. However, up to 7% of DMD mutations are deep intronic and analysis of muscle-derived RNA is an important diagnostic step for patients who have negative genomic testing but abnormal dystrophin expression in muscle. In this study, muscle biopsies were evaluated from 19 patients with clinical features of a dystrophinopathy, but negative clinical DMD mutation analysis. Reverse transcription-polymerase chain reactionor high-throughput RNA sequencingmethods identified 19 mutations with one of three pathogenic pseudoexon types: deep intronic point mutations, deletions or insertions, and translocations. In association with point mutations creating intronic splice acceptor sites, we observed the first examples of DMD pseudo 3'-terminal exon mutations causing high efficiency transcription termination within introns. This connection between splicing and premature transcription termination is reminiscent of U1 snRNP-mediating telescripting in sustaining RNA polymerase II elongation across large genes, such as DMD. We propose a novel classification of three distinct types of mutations identifiable by muscle RNA analysis, each of which differ in potential treatment approaches. Recognition and appropriate characterization may lead to therapies directed toward full-length dystrophin expression for some patients.

Therapeutic exon skipping as a treatment for Duchenne muscular dystrophy (DMD) has largely concentrated on the delivery of antisense oligomers to treat out-of-frame exon deletions. Here we report o...

Duchenne muscular dystrophy (DMD) is an X-linked progressive disease characterized by loss of dystrophin protein that typically results from truncating mutations in the DMD gene. Current exon-skipping therapies have sought to treat deletion mutations that abolish an open reading frame (ORF) by skipping an adjacent exon, in order to restore an ORF that allows translation of an internally deleted yet partially functional protein, as is seen with many patients with the milder Becker muscular dystrophy (BMD) phenotype. In contrast to that approach, skipping of one copy of a duplicated exon would be expected to result in a full-length transcript and production of a wild-type protein. We have developed an adeno-associated virus (AAV)-based U7snRNA exon-skipping approach directed toward exon 2, duplications of which represent 10% of all DMD duplication mutations. Deletion of exon 2 results in utilization of an exon 5 internal ribosome entry site (IRES) that allows translation beginning in exon 6 of a highly protective dystrophin protein, providing a wide therapeutic window for treatment. Both intramuscular and systemic administration of this vector in the Dup2 mouse model results in robust dystrophin expression and correction of muscle physiologic defects, allowing dose escalation to establish a putative minimal efficacious dose for a human clinical trial.

Exon skipping therapies for Duchenne muscular dystrophy that restore an open reading frame can be induced by the use of non-coding U7 small nuclear RNA (U7snRNA) modified by an antisense exon-targeting sequence delivered by an adeno-associated virus (AAV) vector. We have developed an AAV vector (AAV9.U7-ACCA) containing four U7snRNAs targeting the splice donor and acceptor sites of dystrophin exon 2, resulting in highly efficient exclusion of DMD exon 2. We assessed the specificity of splice variation induced by AAV9.U7-ACCA delivery in the Dmd exon 2 duplication (Dup2) mouse model via an unbiased RNA-seq approach. Treatment-related effects on pre-mRNA splicing were quantified using local splicing variation (LSV) analysis. Filtering the transcriptome for differences in treatment-related splicing resulted in only 16 candidate off-target LSVs. Only a single candidate off-target LSV was found in both tissues and occurred at a known variable cassette exon. In contrast, four LSVs represented significant on-target correction of Dmd exon 2 splicing and transcriptome analysis showed correction of known dystrophin-deficient gene dysregulation. We conclude that the absence of off-target splicing induced by treatment with the U7-ACCA vector supports the continued clinical development of this approach.

The uridine-rich 7 (U7) small nuclear RNA (snRNA) is a component of a small nuclear ribonucleoprotein (snRNP) complex. U7 snRNA naturally contains an antisense sequence that identifies histone premessenger RNAs (pre-mRNAs) and is involved in their 3' end processing. By altering this antisense sequence, researchers have turned U7 snRNA into a versatile tool for targeting pre-mRNAs and modifying splicing. Encapsulating a modified U7 snRNA into a viral vector such as adeno-associated virus (also referred as vectorized exon skipping/inclusion, or VES/VEI) enables the delivery of this highly efficacious splicing modulator into a range of cell lines, primary cells, and tissues. In addition, and in contrast to antisense oligonucleotides, viral delivery of U7 snRNA enables long-term expression of antisense sequences in the nucleus as part of a stable snRNP complex. As a result, VES/VEI has emerged as a promising therapeutic platform for treating a large variety of human diseases caused by errors in pre-mRNA splicing or its regulation. Here we provide an overview of U7 snRNA's natural function and its applications in gene therapy.

Background Canine models of Duchenne muscular dystrophy (DMD) are a valuable tool to evaluate potential therapies because they faithfully reproduce the human disease. Several cases of dystrophinopathies have been described in canines, but the Golden Retriever muscular dystrophy (GRMD) model remains the most used in preclinical studies. Here, we report a new spontaneous dystrophinopathy in a Labrador Retriever strain, named Labrador Retriever muscular dystrophy (LRMD). Methods A colony of LRMD dogs was established from spontaneous cases. Fourteen LRMD dogs were followed-up and compared to the GRMD standard using several functional tests. The disease causing mutation was studied by several molecular techniques and identified using RNA-sequencing. Results The main clinical features of the GRMD disease were found in LRMD dogs; the functional tests provided data roughly overlapping with those measured in GRMD dogs, with similar inter-individual heterogeneity. The LRMD causal mutation was shown to be a 2.2-Mb inversion disrupting the DMD gene within intron 20 and involving the TMEM47 gene. In skeletal muscle, the Dp71 isoform was ectopically expressed, probably as a consequence of the mutation. We found no evidence of polymorphism in either of the two described modifier genes LTBP4 and Jagged1. No differences were found in Pitpna mRNA expression levels that would explain the inter-individual variability. Conclusions This study provides a full comparative description of a new spontaneous canine model of dystrophinopathy, found to be phenotypically equivalent to the GRMD model. We report a novel large DNA mutation within the DMD gene and provide evidence that LRMD is a relevant model to pinpoint additional DMD modifier genes.

Duchenne muscular dystrophy (DMD) is associated with the loss of dystrophin, which plays an important role in myofiber integrity via interactions with β-dystroglycan and other members of the transmembrane dystrophin-associated protein complex. The ZZ domain, a cysteine-rich zinc-finger domain near the dystrophin C-terminus, is implicated in forming a stable interaction between dystrophin and β-dystroglycan, but the mechanism of pathogenesis of ZZ missense mutations has remained unclear because not all such mutations have been shown to alter β-dystroglycan binding in previous experimental systems. We engineered three ZZ mutations (p.Cys3313Phe, p.Asp3335His, and p.Cys3340Tyr) into a short construct similar to the Dp71 dystrophin isoform for in vitro and in vivo studies and delineated their effect on protein expression, folding properties, and binding partners. Our results demonstrate two distinct pathogenic mechanisms for ZZ missense mutations. The cysteine mutations result in diminished or absent subsarcolemmal expression because of protein instability, likely due to misfolding. In contrast, the aspartic acid mutation disrupts binding with β-dystroglycan despite an almost normal expression at the membrane, confirming a role for the ZZ domain in β-dystroglycan binding but surprisingly demonstrating that such binding is not required for subsarcolemmal localization of dystrophin, even in the absence of actin binding domains.

We report for the first time the characterization of disease-causing exonic rearrangements in the large-sized gene encoding dysferlin. A newly developed kit for multiplex ligation-dependent probe amplification analysis of the dysferlin gene was used for a total of 12 samples from patients with suspected diagnosis of primary dysferlinopathy. This analysis and subsequent genomic quantitative real-time PCR evidenced exonic rearrangements in five patients, including four different exonic deletions and one duplication. Altogether, our findings confirm the existence of exonic rearrangements as disease-causing mutations in primary dysferlinopathies.

We recently identified an internal ribosome entry site (IRES) within exon 5 of the DMD gene. Mutations that truncate the reading frame 5’ of this exon can result in use of the IRES for alternate translational initiation beginning within exon 6 that results in expression of an N-truncated isoform. Despite lacking the calponin homology domain 1 (CH1) of the actin binding domain 1 (ABD1), this isoform is highly functional, as demonstrated by the minimal symptoms in patients who express it. Consistent with genotype-phenotype correlations in DMD patients, the IRES is not active in the presence of exon 2 duplication but is active when exon 2 is deleted. We developed an AAV9.U7snRNA vector to that truncates DMD reading frame by skipping of exon 2, and have shown that in a Duchenne muscular dystrophy (DMD) mouse model carrying a duplication of exon 2 (the Dup2 mouse), postnatal intramuscular (IM) or intravascular (IV) treatment results in functional and pathologic improvement in skeletal muscle. Relevant to efforts to identify and treat DMD patients at an earlier age, we sought here to determine whether earlier gene transfer might slow down or even prevent the development of pathology. Dup2 mice were injected via facial vein at postnatal day 1 (P1) with 1E12 total vector genomes of the AAV9.U7snRNA vector and sacrificed at either 1, 3, 6, or 12 months post-injection for evaluation of exon 2 skipping by RT-PCR, quantification of dystrophin expression, and characterization of histopathology. To model the applicability of this approach beyond exon 2 duplication patients, the same vector was used to treat 6 human patient fibroblast-derived transdifferentiated myoblasts (FibroMyoD cells) harboring various mutations within exons 1 to 4. In the Dup2 mouse, efficient skipping and abundant dystrophin expression were present up to one year following the single AAV injection. Dystrophic pathology was absent at all-time points; at one year, less than 1% of fibers showed central nucleation, in comparison to ~70% in untreated Dup2 mice. Two tests on the ex vivo diaphragm preparations: isometric force (providing assessment of strength), and eccentric contractions (evaluating sarcolemma stability) were performed at 3 and 6 months following P1 injection. Both tests demonstrated little to no difference between treated animals and wild type mice. In all FibroMyoD cultures, abundant exon 2 skipping and dystrophin expression were detected in myotubes at 14 days of culture after treatment. These results suggest that this exon-skipping vector offers a therapeutic approach not only to patients with exon 2 duplications but with all mutations within the first four DMD exons (~6% of patients), an area of the gene largely ignored by the current therapeutic approaches. This work strongly supports the idea that early treatment of these patients will have longstanding and significant benefit resulting in a better outcome.

Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity has been shown to result from alternative translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. Here we demonstrate that this isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid inducible. We confirmed IRES activity by both peptide sequencing and ribosome profiling in muscle from individuals with minimal symptoms despite the presence of truncating mutations. We generated a truncated reading frame upstream of the IRES by exon skipping, which led to synthesis of a functional N-truncated isoform in both human subject-derived cell lines and in a new DMD mouse model, where expression of the truncated isoform protected muscle from contraction-induced injury and corrected muscle force to the same level as that observed in control mice. These results support a potential therapeutic approach for patients with mutations within the 5' exons of DMD.

Most mutations that truncate the reading frame of the DMD gene result in loss of dystrophin expression and lead the severe Duchenne muscular dystrophy. However, frame-truncating mutations within the first five exons of DMD result in mild dystrophinopathy with expression of a N-truncated dystrophin. We have recently shown that this is due to activation of an internal ribosome entry site (IRES) within exon 5 resulting in translation from an exon 6 AUG codon.We demonstrated that this IRES is active in patients expressing the N-truncated dystrophin, raising the possibility of the therapeutic use of this isoform. To explore this we developed a novel out-of-frame exon-skipping approach that uses AAV-mediated U7snRNA to efficiently skip exon 2. By injecting this AAV vector into a DMD mouse model carrying a duplication of exon 2 (Dup2), this generates a truncated reading frame, leading to activation of the IRES and synthesis of the N-truncated isoform.We now demonstrate that despite lacking the first half of the canonical actin binding domain 1, this N-truncated protein is highly functional. Intramuscular injection of the AAV1.U7snRNA vector into Dup2 mice results in high levels of expression of the N-truncated isoform by 4 to 6 weeks post-injection, along with complete correction of the physiologic and pathologic features as measured by Evans blue dye uptake, hindlimb grip strength, tibialis anterior specific force, and force correction after eccentric contraction. Preliminary results supports that systemic delivery of AAV9.U7snRNA vector into Dup2 mice induce expression of this functional isoform into all muscle including heart and diaphragm, thereby improving muscle histopathology.Following treatment, a genome-wide normalized RPF-Seq data analysis (Ribosome Protected Fragment) was performed to check if the treatment restored the Haslett gene lists (gene altered in DMD) to a ‘non-dystrophic’ pattern. Our data clearly indicates that the treatment restored the global expression pattern to a more normal pattern. This level of correction to that of control mice supports the idea that this novel therapeutic approach should be beneficial for the 6% of patients with mutations within the first five exons of DMD.

Currently, exon skipping therapies for Duchenne muscular dystrophy (DMD) have been developed for patients with out-of-frame deletions where treatment will lead translation of an internally truncated but partially functional dystrophin protein. In contrast, we are focusing on treating duplications mutations, accounting for around 6% of all mutations, resulting in wild-type transcript and a full-length protein. Modeling the most common single exon duplication we have developed the first duplication mouse containing a duplicated exon 2. We have performed a proof-of-principle study using antisense oligomer (AO)-induced exon 2 skipping using this Dup2 mouse. Intramuscular (TA) injections of exon 2-directed different antisense peptide-morpholino conjugates (PPMO) were performed at either 10 or 20 ug total PPMO (N=6 muscles each) doses. Mice were injected at 8 weeks and sacrificed 1 month later for muscle analysis of DMD mRNA and dystrophin protein expression. Additionally, systemic (tail vein) injections are being conducted of at a dose of 30 ug/kg, at 1 week, 2 week and 1 month timepoints. For the IM studies, a gradient of exon 2 exclusion was seen by RT-PCR at both doses with corresponding high levels of properly localized dystrophin protein by IF and western blot.Analyses of RT-PCR and protein expression are underway for the systemic delivery PPMOs. These data suggest that skipping of a duplicated exon 2 may be a feasible therapeutic approach, particularly because skipping of exon 2 may be associated with an apparently unlimited therapeutic window. Over-skipping - to the exclusion of exon 2 entirely - results in activation of an internal ribosome entry site (IRES) located in exon 5 of dystrophin that allows for cap-independent translation from an alternative initiation site within exon 6. This alternate dystrophin isoform is highly functional despite being N-truncated, consistent with the observation that patients expressing it have minimally symptomatic (or even asymptomatic) Becker muscular dystrophy (BMD), and suggesting a potential route to therapy for any of the approximately 5% of patients with mutations in the 5’ end of the gene.

International audience; no abstract

International audience; Mutations in the dysferlin gene (DYSF) lead to a complete or partial absence of the dysferlin protein in skeletal muscles and are at the origin of dysferlinopathies, a heterogeneous group of rare autosomal recessive inherited neuromuscular disorders. As a step towards a better understanding of the DYSF mutational spectrum, and towards possible inclusion of patients in future therapeutic clinical trials, we set up the Universal Mutation Database for Dysferlin (UMD-DYSF), a Locus-Specific Database developed with the UMD (R) software. The main objective of UMD-DYSF is to provide an updated compilation of mutational data and relevant interactive tools for the analysis of DYSF sequence variants, for diagnostic and research purposes. In particular, specific algorithms can facilitate the interpretation of newly identified intronic, missense- or isosemantic-exonic sequence variants, a problem encountered recurrently during genetic diagnosis in dysferlinopathies. UMD-DYSF v1.0 is freely accessible at www.umd.be/DYSF/. It contains a total of 742 mutational entries corresponding to 266 different disease-causing mutations identified in 558 patients worldwide diagnosed with dysferlinopathy. This article presents for the first time a comprehensive analysis of the dysferlin mutational spectrum based on all compiled DYSF disease-causing mutations reported in the literature to date, and using the main bioinformatics tools offered in UMD-DYSF.

We read with interest but also some surprise, the recent ‘Therapeutic exon skipping for Dysferlinopathies?' article by Aartsma-Rus et al,1 published in the Eur J Hum Genet (advance online publication, 10 February 2010; doi:10.1038/ejhg.2010.4). This report contains some inaccuracies and mistakes, and we do not agree with some of its main contents. Therefore, for the sake of the scientific and patients communities, we consider it essential to point out and discuss some of the results and subsequent conclusions appearing in the ‘results and discussion' section, as well as in figures and tables. Antisense-mediated exon skipping constitutes a relevant therapy for several genetic diseases associated with premature termination codons (PTCs) or frameshift mutations lying in dispensable in frame exons. However, knowledge of the specific targeted gene, mRNA and resulting protein, is, among others, an essential pre-requisite toward designing pertinent exon-skipping strategies. In the report by Aartsma-Rus et al,1 it appears that several of these pre-requisites have been overlooked. Here we intend to discuss and contradict some important assertions and results that appear in both the text and the figures of the article by Aartsma-Rus et al,1 and place our comments in the perspective of a recently published report from our group on the same matter.2

International audience; Mutations in DYSF encoding dysferlin cause primary dysferlinopathies, autosomal recessive diseases that mainly present clinically as Limb Girdle Muscular Dystrophy type 213 and Miyoshi myopathy. More than 350 different sequence variants have been reported in DYSE. Like dystrophin, the size of the dysferlin mRNA is above the limited packaging size of AAV vectors. Alternative strategies to AAV gene transfer in muscle cells must then be addressed for patients. A gene therapy approach for Duchenne muscular dystrophy was recently developed, based on exon-skipping strategy. Numerous sequences are recognized by splicing protein complexes and, when specifically blocked by antisense oligoucleotides (AON), the corresponding exon is skipped. We hypothesized that this approach could be useful for patients affected with dysferlinopathics. To confirm this assumption, exon 32 was selected as a prioritary target for exon skipping strategy. This option was initially driven by the report from Sinnreich and colleagues of a patient with a very mild and late-onset phenotype associated to a natural skipping of exon 32. Three different antisense oligonucleotides were tested in myoblasts generated from control and patient MyoD transduced fibroblasts, either as oligonucleotides or after incorporation into lentiviral vectors. These approaches led to a high efficiency of exon 32 skipping. Therefore, these results seem promising, and could be applied to several other exons in the DYSF gene. Patients carrying mutations in exons whose the in-frame suppression has been proven to have no major consequences on the protein function, might benefit of exon-skipping based gene correction.

To date, exon-skipping therapies for Duchenne muscular dystrophy (DMD) patients have focused on patients with out-of-frame exon deletions, in whom treatment results in larger but in-frame internal deletions which lead to translation of an internally truncated but partially functional dystrophin protein. We are developing exon-skipping therapies for exon duplication mutations, accounting for around 6% of all mutations, with the intent to induce production of wild-type transcripts and protein. As a model of the most common single exon duplication, we have developed a new mouse with a duplication of exon 2 (the Dup2 mouse) that largely recapitulates the findings in the standard mdx mouse. Using this mouse, we are testing both virally (AAV) mediated skipping induced by a modified U7snRNA (rAAV9.U7.ACCA), and antisense oligomer-induced skipping. Intramuscular injections of the tibialias anterior (TA) (N=6 muscles each) were performed rAAV9.U7.ACCA at 6 doses between 1×1010 and 1×1012 total vector genomes. Intramuscular (TA) injections of an exon 2-directed antisense peptide-morpholino conjugate (PPMO) were performed at doses of either 10 or 20 ug total PPMO (N=3 each). For each study, mice were sacrificed 1 month post injection and muscle analysis of DMD mRNA and dystrophin protein expression. Treatment with either modality results in significant skipping of the duplicated exon 2, and expression of a functional dystrophin isoform at levels of up to 30% of normal with AAV-mediated skipping. Physiology has been assessed in the AAV-treated mice, in which correction of TA absolute force deficits in comparison to the background Bl6 strain are seen, along with a partial yet significant correction of eccentric contraction injury in comparison to untreated Dup2 mice. These data suggest that skipping of a duplicated exon 2 may be a feasible therapeutic approach, particularly because skipping of exon 2 may be associated with an apparently unlimited therapeutic window. Over-skipping – to the exclusion of exon 2 entirely – results in activation of an internal ribosome entry site (IRES) located in exon 5 of dystrophin that allows for cap-independent translation from an alternative initiation site within exon 6. This alternate dystrophin isoform is highly functional despite being N-truncated, consistent with the observation that patients expressing it have minimally symptomatic (or even asymptomatic) Becker muscular dystrophy (BMD), and suggesting a potential route to therapy for any of the approximately 5% of patients with mutations in the 5’ end of the gene.

Nonsense mutations within exon 1 of DMD do not result in severe DMD but instead lead to very mild BMD, due to an alternative initiation of translation at AUG codons within in exon 6. This leads to translation of a nearly full-length but N-terminal truncated dystrophin lacking the first half of the canonical actin binding domain 1 (ABD1). We have identified the motif encoded in exon 5 that recruits ribosomes for alternate translational initiation within exon 6, and using a dual luciferase reporter system we have determined that this motif is selectively activated in muscle cell lines but not fibroblasts or HEK cells. Our data suggest that this motif is an IRES (internal ribosome entry site) as complementary experiments have ruled out any promoter activity or aberrant splicing. The exceedingly mild clinical features of patients with an exon 1 DMD founder allele (p.Trp3X) suggest that the product of this IRES initiation is a highly functional protein. We are therefore exploring different strategies to induce IRES utilisation for therapeutic purposes in patients with mutations in exons 1 through 4. One of them is based on forced synthesis of this IRES protein isoform that can be induced by exon 2 skipping. This leads to a frameshift and premature stop codon in exon 3, which thereby force the use of the IRES. We have developed four different antisense sequences for efficient skipping of exon 2 that are incorporated into a short U7 RNA derivative that was previously used in other studies to induce efficient exon skipping in DMD . We are currently testing these antisense constructs in patient cells carrying mutations in the first exons to evaluate the potential positive benefit of this out of frame skipping strategy.

Frame-truncating mutations within the first five exons of the DMD gene typically do not result in Duchenne muscular dystrophy, but instead result in milder dystrophinopathy syndromes. This was first demonstrated in individuals carrying the c.9G > A (p.Trp3X) mutation, a North American founder allele associated with clinical severity ranging from a very mild Becker muscular dystrophy phenotype to a complete absence of symptoms. We have previously shown that the mild phenotype associated with this and similar 5’ exon mutations is due to alternative initiation of translation from methionines encoded in exon 6, a process mediated by the presence of a muscle-specific and glucocorticoid-inducible internal ribosomal entry site (IRES) found within exon 5. The resultant N-truncated dystrophin protein lacks the first calponin homology domain of the canonical actin binding domain 1. Nevertheless, it is highly functional, raising the possibility of the therapeutic use of this isoform. We hypothesized that disruption of the mRNA open reading frame via exon skipping – resulting in a downstream premature termination codon – would stimulate IRES utilization. The feasibility of this approach is supported by the recent identification of an asymptomatic patient harbouring an exon 2 deletion, and we designed U7snRNA vectors targeting exon 2 for use in both patient-derived cell lines and in a new DMD mouse model harbouring an exon 2 duplication. Both in vitro and in vivo we can stimulate IRES activity, and by combining exon-skipping and glucocorticoid treatment we are able to restore expression of a properly localized yet N-truncated dystrophin. These results not only provide evidence for the functionality of the dystrophin IRES – and, hence, for the presence of a novel N-truncated dystrophin isoform under yet to be clarified physiologic conditions – but also suggest that out-of-frame skipping is a promising therapeutic approach for DMD patients harbouring 5’ mutations.

International audience; Dysferlinopathies are autosomal recessive disorders caused by mutations in the dysferlin (DYSF) gene, encoding the dysferlin protein. DYSF mutations lead to a wide range of muscular phenotypes, with the most prominent being Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B) and the second most common being LGMD. Symptoms generally appear at the end of childhood and, although disease progression is typically slow, walking impairments eventually result. Dysferlin is a modular type II transmembrane protein for which numerous binding partners have been identified. Although dysferlin function is only partially elucidated, this large protein contains seven calcium sensor C2 domains, shown to play a key role in muscle membrane repair. On the basis of this major function, along with detailed clinical observations, it has been possible to design various therapeutic approaches for dysferlin-deficient patients. Among them, exon-skipping and minigene transfer strategies have been evaluated at the preclinical level and, to date, represent promising approaches for clinical trials. This review aims to summarize the pathophysiology of dysferlinopathies and to evaluate the therapeutic potential for treatments currently under development. 2011 The Feinstein Institute for Medical Research, www.feinsteininstitute.org Online address: http://www.molmed.org doi: 10.2119/molmed.2011.00084